The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca2+, basal SACY activity is suppressed by Ca2+. Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo.DOI: http://dx.doi.org/10.7554/eLife.14052.001

Navigation of sperm in fluid flow, called rheotaxis, provides long‐range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+) influx via the sperm‐specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark‐field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper‐deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+‐signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux.

Delineating tumor margins as accurately as possible is of primordial importance in surgical oncology: extent of resection is associated with survival but respect of healthy surrounding tissue is necessary for preserved quality of life. The real-time analysis of the endogeneous fluorescence signal of brain tissues is a promising tool for defining margins of brain tumors. The present study aims to demonstrate the feasibility of multimodal optical analysis to discriminate fresh samples of gliomas, metastases and meningiomas from their appropriate controls. Tumor samples were studied on an optical fibered endoscope using spectral and fluorescence lifetime analysis and then on a multimodal set-up for acquiring spectral, one and two-photon fluorescence images, second harmonic generation signals and two-photon fluorescence lifetime datasets. The obtained data allowed us to differentiate healthy samples from tumor samples. These results confirmed the possible clinical relevance of this real-time multimodal optical analysis. This technique can be easily applied to neurosurgical procedures for a better delineation of surgical margins.

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.